Improving the maturation of human pluripotent stem cell - derived hepatocyte-like cells using an automated-design of experiments approach

Human primary hepatocytes (pHEPs) are used as the gold standard model for drug screening and toxicity testing in the drug development pipeline. However, the rapid post isolation changes in cell structure, morphology, gene expression and metabolic activity, together with donor availability and heterogeneity, limit their utility. Directed differentiation of human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) into hepatocyte-like cells (HLCs) can be achieved within a short time and in unlimited quantities; however their metabolic activity closely represents foetal hepatocytes but not an adult hepatocyte metabolic profile. Studies have identified compounds that can increase expression of some adult enzymes, but a systematic evaluation of multiple compounds in a range of concentrations has never been reported in human cells. Studying the maturation of HLCs requires identification of distinct markers that are only expressed in the adult liver cells to clearly evaluate maturation characteristics as the current literature predominantly investigates the expression of the characteristics observed during the foetal stages. In this thesis, the current status of the field is reviewed in Chapter 1. The existing hepato-cyte differentiation protocol has been re-designed in Chapter 3 taking into consideration up-to-date methods, cell line differences and culture medium, availability of human Plu-ripotent Stem Cells (hPSCs) and differentiation into HLCs on a TECAN Freedom Evo 200 automated cell culture robot. Based on a published proteomic study, a quantifiable high-throughput assay was developed to identify distinct adult enzyme markers Cytochrome 450 1A2 (CYP450 1A2), Cytochrome 450 2C9 (CYP450 2C9) and Cytochrome 450 2A6 (CYP450 2A6) in Chapter 4. Finally, Design of Experiment (DoE) approaches were utilised in Chapter 5 to screen 46 identified candidate compounds at a range of concentrations. The results predicted that SR12813 at 1.5uM, taurocholate acid at 155uM, CHIR 99021 at 8.5uM, all-trans retinoic acid at 3uM and ascorbic acid at 1.5mM can enhance the expression of CYP2C9. Chapter 6 discusses the advances of this thesis and their relevance to the current literature and the HLCs field